AP4A MODULATION PC12 CELL MONOAMINE OXIDASE ACTIVITY

AP4A 调节 PC12 细胞单胺氧化酶活性

• 批准号: 2274611
• 负责人: EDWARD B PIVORUN
• 金额: $ 10.95万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2274611
• 关键词: PC12 cells; SDS polyacrylamide gel electrophoresis; adenine nucleotides; adenylate cyclase; amine oxidase (flavin); cyclic AMP; enzyme activity; enzyme linked immunosorbent assay; excitatory aminoacid; guanosinetriphosphatases; high performance liquid chromatography; laboratory rat; protein tyrosine kinase; protein tyrosine phosphatase; putamen; synaptosomes

DESCRIPTION: (from Abstract) Most studies dealing with monoamine oxidase (MAO) have emphasized the development of inhibitors of this enzyme for controlling neurotransmitter metabolism in patients with diseases such as Parkinson's. The literature is replete with information on natural and man-made inhibitors of MAO activity. However, there is virtually no information on modulators of MAO that result in enhanced activity of this enzyme. The applicants' laboratory has recently demonstrated that diadenosine tetraphosphate, Ap4A, may be one, of only a few, endogenous extracellular stimulatory agents of MAO activity. They have undertaken a series of experiments aimed at understanding the signal transduction pathway associated with Ap4A receptor binding and the enhanced activation of MAO. An understanding of the transduction pathway(s) involved with Ap4A receptor activation may result in a better understanding of the signal transduction events associated with MAO activation. In addition, if the Ap4A activated transduction pathway(s) is a novel activation pathway(s) for MAO, knowledge of this pathway(s) may allow for the development of a new class of agonists, antagonists or pharmacological agents for controlling MAO activity. Abnormal or elevated level of Ap4A in the CNS and adrenal gland may lead to pathogenic conditions associated with depleted catecholamine stores, such as Parkinson's Disease. The potential importance of MAO in the catabolism of various xenobiotics suggests that Ap4A may have an important therapeutic role. The specific aims of this proposal are: (A) To determine the dosage effects of the extracellular diadenosine polyphosphates on MAO activity in PC12 cells and brain synaptosomes obtained form the caudate putamen of rat brains. Dopamine metabolites will be measured by using 1) HPLC with electrochemical detection and 2) radiochemical analyses. The temporal dynamics of the application of Ap4A will be determined to assess the effects of short term and chronic exposure of the cells and synaptosomes; (B) To determine the effects of extracellular Ap4A on adenylate cyclase activity and cAMP levels in PC12 cells. cAMP levels will be assayed by an enzyme linked immunoassay; adenylate cyclase activity will be determined using [a32P] ATP and chromatography; (C) To determine the role of G protein involvement in the Ap4A elicited response by measuring GTPase enzymatic activity; (D) To determine the involvement of tyrosine kinases and tyrosine phosphatases in the Ap4A initiated signal transduction pathway. SDS-PAGE and [32P]phosphate will be used to label cells and determine the extent of phosphorylation or dephosphorylation of specific proteins. (E) To determine the effects of Ap4A delivery to the caudate putamen of the conscious rat with in vivo microdialysis in conjunction with HPLC.

描述：（来自摘要）大多数涉及单胺的研究 氧化酶（MAO）强调了抑制剂的发展 这种用于控制患者神经递质代谢的酶 帕金森氏症等疾病。 文学充满了 有关毛泽东活动的自然和人为抑制剂的信息。 但是，几乎没有关于MAO调制器的信息 导致该酶的活性增强。 申请人实验室 最近证明了二甲二磷酸二磷酸AP4A可能是 其中之一只有少数几个内源性的细胞外刺激剂 毛活动。 他们进行了一系列针对的实验 了解与AP4A相关的信号转导途径 受体结合和MAO的激活增强。 一种理解 与AP4A受体激活有关的转导途径的 可以更好地了解信号转导事件 与MAO激活相关。 另外，如果AP4A激活 转导途径是Mao的新型激活途径， 对这一途径的了解可能允许发展新的 激动剂，拮抗剂或药理学剂的类别 控制MAO活动。 AP4A的异常或升高 中枢神经系统和肾上腺可能导致致病条件 随着枯竭的儿茶酚胺商店，例如帕金森氏病。 这 毛的潜在重要性在各种分解代谢中 异种生物表明，AP4A可能具有重要的治疗作用。 该提案的具体目的是：（a）确定剂量 细胞外折叠蛋白多磷酸盐对MAO活性的影响 在PC12细胞和获得的脑突触体中，形成了尾状壳 大鼠大脑。 多巴胺代谢物将通过1）HPLC进行测量 与电化学检测和2）放射化学分析。 这 AP4A应用的时间动力学将确定为 评估细胞短期和长期暴露的影响 和突触体； （b）确定细胞外的影响 PC12细胞中腺苷酸环化酶活性和cAMP水平的AP4A。 将酶连接的免疫测定法测定营地级别；腺苷酸盐 使用[A32P] ATP和色谱法确定环化酶活性； （c）确定G蛋白参与在AP4A中的作用 通过测量GTPase酶活性引起反应； （d）到 确定酪氨酸激酶和酪氨酸的参与 AP4A中的磷酸酶启动信号转导途径。 SDS-PAGE和[32p]磷酸盐将用于标记细胞并确定 特定的磷酸化或去磷酸化的程度 蛋白质。 （e）确定AP4A递送到 有意识大鼠的尾状壳，并在体内微透析中 与HPLC结合。